JPWO2018168579A1 - Genome stability enhancer - Google Patents

Abstract

Disclosed is a genomic stability enhancer containing as an active ingredient at least one selected from the group consisting of enzymatically degraded royal jelly, gnetum or an extract thereof, and gnetin C.

Description

  The present invention relates to a genome stability enhancer.

  Most normal cells stop growing after a certain number of divisions. Resting cells are protected from transformation as long as genomic stability is maintained. This quiescent state is formed with a decrease in histone H2AX levels under the control of the ARF / p53 pathway. A similar cell state in which growth has been stopped has been widely observed in cells constituting organs that normally maintain homeostasis. When the H2AX level is reduced, the cells stop growing and at the same time have a reduced ability to repair DNA damage. Therefore, in cells that have stopped growing, genomic instability is induced due to DNA replication stress caused by excessive growth stimulation. On the other hand, such cells have a mechanism for activating the repair ability associated with transient H2AX induction.

  Histone H2AX is important for the localization of DNA damage repair factors to damaged sites. When double-strand breaks occur in DNA, the surrounding H2AX is phosphorylated. Many DNA damage repair factors interact with this phosphorylated H2AX (called γ-H2AX) to localize at the site of injury. Therefore, by detecting γ-H2AX by immunostaining or the like, it can be used as a marker for DNA damage.

  For example, in Non-Patent Documents 1 and 2, in order to examine the effectiveness of resveratrol against cancer cells, it was confirmed that resveratrol induced DNA damage by detecting γ-H2AX. I have. As a result of DNA damage of cancer cells, an effect of inducing apoptosis is obtained.

  Thus, Non Patent Literatures 1 and 2 describe detection of phosphorylated H2AX, and the expression level of H2AX itself has not been studied. As described above, by transiently inducing H2AX, the ability to repair DNA can be activated and genomic stability can be increased.

  Non-patent Document 3 reports that "DNA repair associated with transient H2AX expression" in "normal cells whose growth has been stopped" was found by treatment with resveratrol and royal jelly.

  Non-Patent Document 4 reports that Factor X involved in DNA repair is transiently induced by resveratrol.

  It has been reported that melinjo, which is rich in royal jelly and resveratrol derivatives, exhibits an anticancer effect, an anti-aging effect, a life extension effect, and the like (Patent Documents 1 and 2, and Non-Patent Documents 5-8).

  Further, no effective treatment method has been found for xeroderma pigmentosum, Huntington's disease, Werner's syndrome, Bloom's syndrome, Lynch's syndrome, etc., which are diseases caused by mutations in the gene repair mechanism.

Japanese Patent No. 5923699 Japanese Patent No. 5979810

Carsinogenesis (2011) 32 (1): 93-101 Ann Hematol (2011) 90: 173-183 Kenichi Yoshioka, 23-C-10 Molecular mechanism study of "genomic instability, mutation, cell transformation" induced by and linked to carcinogenesis-induced stress, Internet <http://crdb.ncc.go.jp/ search / DRTV050.action? rpno = 012013007100000> Kenichi Yoshioka, Hitoshi Shirakawa, Studies on the effects of nutritional components on the prevention of cancer and aging and on the regulation of genomic stability, Internet <http://www.mishima-kaiun.or.jp/assist/docs/SJNo2-yoshioka ,. pdf> Pharmaceutical Journal of Japan (Folia pharmacol. Japon.) (1987) 89: 73-80 Cancer Medicine (2015) 4 (11): 1767-1780 PLoS One (2011) 6 (8): e23527 Bioscience, Biotechnology, and Biochemistry (2015) 79 (12): 2044-2049

  An object of the present invention is to provide a genomic stability enhancer having an excellent genomic stability enhancing action.

  The present inventors have conducted intensive studies to achieve the above object, and found that enzymatically decomposed royal jelly has a higher H2AX-inducing effect than unenzymatically decomposed royal jelly, and that the Melinjo seed extract and the netin C are trans H2AX-inducing action was higher than that of resveratrol.

  The present invention has been completed based on these findings and further studied, and provides the following genomic stability enhancer.

Item 1. A genomic stability enhancer comprising as an active ingredient at least one selected from the group consisting of enzymatically degraded royal jelly, gnetum or an extract thereof, and gnetin C.
Item 2. Item 4. The genomic stability enhancer according to Item 1, which is a cosmetic, a food or drink, a drug, or a quasi drug.
Item 3. A method for enhancing genomic stability, comprising a step of administering to a mammal at least one effective amount selected from the group consisting of enzymatically degraded royal jelly, gnetum or an extract thereof, and gnetin C.
Item 4. Use of at least one selected from the group consisting of enzymatically degraded royal jelly, gnetum or an extract thereof, and gnetin C in the production of a genome stability enhancer.
Item 5. Item 5. The use according to Item 4, wherein the genome stability enhancer is a cosmetic, a food or drink, a drug, or a quasi drug.
Item 6. A composition for enhancing genomic stability comprising as an active ingredient at least one selected from the group consisting of enzymatically degraded royal jelly, gnetum or an extract thereof, and gnetin C.
Item 7. Item 7. The composition for enhancing genome stability according to Item 6, which is a cosmetic, a food or drink, a drug, or a quasi drug.

  Enzymatically degraded royal jelly, gnetum or an extract thereof, and gnetin C have an excellent H2AX expression inducing action, and thus are useful as active ingredients of a genomic stability enhancer.

  Since the genomic stability enhancer of the present invention has an excellent genomic stability enhancing action, it is a disease associated with genomic instability (abnormal gene repair mechanism), xeroderma pigmentosum, Huntington's disease , Werner syndrome, Bloom syndrome, Lynch syndrome and the like are expected to be effective in preventing the onset and alleviating the symptoms.

FIG. 2 shows photographs (upper) showing the results of Western analysis of H2AX expression in Test Example 1, and graphs (lower) showing H2AX expression levels (relative values). Left: untreated royal jelly, right: enzymatically degraded royal jelly FIG. 8 shows photographs (upper) showing the results of Western analysis of H2AX expression in Test Example 2, and graphs (lower) showing H2AX expression levels (relative values). Left: Trans resveratrol, Right: Merinjo FIG. 9 shows photographs (upper) showing the results of Western analysis of H2AX expression in Test Example 3, and a graph (lower) showing H2AX expression levels (relative values). Left: Trans resveratrol, Middle: Merinjo, Right: Gunetin C 9 is a graph showing the survival rate of Msh2 homo- and hetero knockout mice in Test Example 4. ◆: Msh2 (-/-) normal diet n = 11, ■: Msh2 (-/-) diet supplemented with 0.3% Melinjo seed extract n = 8, ▲: Msh2 (+/-) normal diet n = 10, ×: diet supplemented with Msh2 (+/-) 0.3% Melinjo seed extract n = 10 9 is a graph showing the number of tumors formed in the intestinal tract of Msh2 knockout mice in Test Example 5. +: Melinjo seed extract administration group,-: Control group

  Hereinafter, the present invention will be described in detail.

  In this specification, the term “comprise” includes the meaning of “essentially consist of” and the meaning of “consist of”.

  The genomic stability enhancer of the present invention is characterized by containing, as an active ingredient, at least one selected from the group consisting of enzymatically degraded royal jelly, gnetum or an extract thereof, and gnetin C.

  Royal jelly is a milky white jelly-like substance made by mixing secretions secreted from the hypopharyngeal gland and the wow gland by worker bees aged 3 to 12 among bees. The main physiologically active components in royal jelly include, for example, organic acids such as 10-hydroxydecenoic acid (hereinafter referred to as "decenoic acid") unique to royal jelly, proteins, lipids, sugars, vitamins B, Vitamins such as folic acid, nicotinic acid, and pantothenic acid, and various minerals. As the physiological activity and pharmacological activity of royal jelly, antibacterial activity, immunopotentiating activity, antidepressant activity, antitumor activity, anti-inflammatory activity, blood flow increasing effect, etc. are known. In addition, it has been reported that the anticancer drug reduces the side effects and prolongs the life in the event of radiation injury.

  The royal jelly used for the production of enzymatically decomposed royal jelly is not particularly limited, and it is possible to use raw royal jelly, royal jelly powder obtained by drying raw royal jelly and powdered, or raw royal jelly extracted with water or hydrous ethanol. it can.

  The locality of royal jelly is not limited, and may be any of Japan, China, Brazil, European countries, Oceania countries, the United States, and the like.

  The enzyme-degraded royal jelly targeted by the present invention is obtained by treating royal jelly with a protease. Preferably, it is a low-allergen-enzymatically degraded royal jelly, in which allergic reactions caused by proteins contained in royal jelly are suppressed by peptidase treatment. Therefore, the enzymatically degraded royal jelly of the present invention may contain the above-mentioned organic acids such as decenoic acid, lipids, saccharides, vitamins, and various minerals, in addition to the peptidase hydrolyzate of the protein contained in the royal jelly.

  As the enzyme used for enzymatically decomposing royal jelly, peptidase can be preferably mentioned. The peptidase used may have at least one of an endopeptidase action and an exopeptidase action, and is preferably a substance having at least an endopeptidase action, more preferably a substance having both of these actions.

  The enzymatically degraded royal jelly of the present invention is preferably a substance which is reduced in allergen by hydrolyzing a protein contained in royal jelly. For this purpose, it is preferable to hydrolyze royal jelly using a peptidase having at least an endopeptidase action (endopeptidase), preferably using a peptidase having both an endopeptidase action and an exopeptidase action. Here, as a peptidase having both an endopeptidase action and an exopeptidase action, a peptidase having both these actions at the same time may be used alone, or a peptidase having an endopeptidase action (endopeptidase) and an exopeptidase may be used. It may be used in combination with a peptidase having an action (exopeptidase).

  As the endopeptidase that can be used in the present invention, any endopeptidase may be used as long as it is a proteolytic enzyme having at least endopeptidase activity. For example, endopeptidases of animal origin (e.g., trypsin, chymotrypsin, etc.), plants (e.g., papain, etc.), or microorganisms (e.g., lactic acid bacteria, yeast, mold, Bacillus subtilis, actinomycetes, etc.) can be widely exemplified. it can.

  The exopeptidase may be any proteolytic enzyme having at least exopeptidase activity. For example, carboxypeptidase, aminopeptidase, or exopeptidase derived from a microorganism (eg, lactic acid bacterium, Aspergillus genus, Rhizopus bacterium, etc.) or pancreatin and pepsin, which also have endopeptidase activity, can be exemplified.

  Incidentally, peptidases include exopeptidases having substantially only exopeptidase action, endopeptidases having substantially only endopeptidase action, and peptidases having both exopeptidase action and endopeptidase action. Among these, enzymes having both an exopeptidase action and an endopeptidase action can be used as `` endopeptidase '' when the endopeptidase action is strong, and `` exopeptidase '' when the exopeptidase action is strong. When the exopeptidase action and the endopeptidase action are equivalent or almost equivalent, it can be used as a peptidase having both the endopeptidase action and the exopeptidase action at the same time.

  Among such peptidases, preferred examples of enzymes having an exopeptidase activity include, for example, Aspergillus orizae-producing peptidases (trade names: Horsemizyme G, Promod 192P, Promod 194P, Sumiteam FLAP), Aspergillus soe (Aspergillus sojae) -produced peptidase (trade name: Sternzyme B15024), Aspergillus-produced peptidase (trade name: coclase P), Rhizopus oryzae-produced peptidase (trade name: peptidase R), and the like.

  Preferred examples of peptidases having an endopeptidase activity include, for example, peptidases produced by Bacillus subtilis (Bacillus subtilis) (trade name: Orientase 22BF, Nucleysin), peptidases produced by Bacillus licheniformis (Bacillus licheniformis) Name: Alcalase), Bacillus stearothermophilus-producing peptidase (trade name: Protease S), Bacillus amyloliquefaciens-producing peptidase (trade name: Neutrase), Bacillus genus-producing peptidase (Product name: Protamex).

  Furthermore, preferred examples of peptidases having both exopeptidase activity and endopeptidase activity, particularly alkaline peptidases, include, for example, Streptomyces griseus (Streptomyces griseus) -producing peptidase (trade name: Actinase AS), Aspergillus oryzae (Aspergillus oryzae). orizae) -producing peptidase (trade name: protease A, flavorzyme), Aspergillus melleus-producing peptidase (trade name: protease P), and the like. According to the enzyme treatment using such a peptidase, the protein can be reduced in molecular weight by one-step enzyme treatment, so that the operation is simple and the disappearance and significant reduction of the physiological activity of the useful component contained in the royal jelly can be achieved. There is an advantage that it can be prevented.

  The amount of peptidase used for royal jelly varies depending on the royal jelly concentration, enzyme titer, reaction temperature and reaction time to be used, but in general, peptidase is used in a ratio of 50 to 10,000 action units per 1 g of protein contained in royal jelly. Is preferred. At this time, the peptidase may be added to the royal jelly all at once, or may be added in small portions.

  During the peptidase treatment, the pH of the royal jelly is selected from the range of pH 2 to 12, preferably pH 7.5 to 10, more preferably pH 7.8 to 9, corresponding to the optimum pH of the enzyme used. Specifically, before the peptidase is added to the royal jelly, an acid such as pH 2 to 12, preferably pH 7.5 to 10, more preferably pH 7.8 to 9, depending on the type of the enzyme used, The pH is adjusted to a desired value by adding an alkali agent or a buffer. In this case, as the acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid and the like; as the alkaline agent, sodium hydroxide, potassium hydroxide, potassium carbonate and the like; and as the buffering agent, a phosphate buffer, A citrate buffer and the like can be exemplified.

  The temperature of the peptidase treatment is not particularly limited, and includes a peptidase action, preferably an endopeptidase action, more preferably an optimal temperature range in which both actions of the endopeptidase action and the exopeptidase action are expressed, a range that can be put to practical use, That is, it is usually selected from the range of 30 to 70 ° C. By maintaining the temperature at a temperature lower or higher than the optimum temperature of the peptidase (preferably about 40 to 50 ° C), for example, in the range of 50 to 60 ° C, spoilage in the peptidase treatment step can also be prevented. The duration of the peptidase treatment depends on the type of the enzyme used and the reaction conditions such as the reaction temperature and pH, and is not particularly limited.

  In addition, the royal jelly can be subjected to proteolytic enzyme treatment as it is or in a state of being dissolved or dispersed in water. When these are in a dry form, they are preferably subjected to proteolytic enzyme treatment in a state of being dissolved in water.

  Termination of the peptidase treatment is performed by inactivating or removing the peptidase. The deactivation operation can be performed by a heat treatment (for example, at 85 ° C. for 15 minutes).

  The enzymatically degraded royal jelly in the present invention may be at least royal jelly treated with peptidase as described above.In addition to peptidase treatment, royal jelly treated with glycolytic enzymes in combination with other enzymes, for example, peptidase treatment. Is also included.

  As the enzymatically decomposed royal jelly in the present invention, a dried and powdered royal jelly after enzymatic decomposition can also be used. Known drying methods employed in general food processing, such as ventilation drying, natural drying such as sun drying, forced drying by heating with electricity or the like, fluidized bed drying, spray drying, high frequency drying, freeze drying, etc. A method can be used, preferably lyophilization. As a pulverization method for pulverization, a known method such as a method of pulverization using a pulverizer (mill) (for example, a pin mill, a hammer mill, a ball mill, a jet mill, a roller mill, a colloid mill, etc.) can be used.

  Gnetsum (also known as Merinjo, scientific name: Gnetum gnemon L., English name: Gnemon tree, Indonesian name: Melinjo, Belinjo) is a plant belonging to the family Gnetum and is cultivated in Southeast Asia.

  In the present invention, the site where the gnetum is used is not particularly limited, and includes fruits (or seeds), flowers, leaves, and the like, and is preferably seeds. In addition, processed materials can also be used as the guntum, and examples of such processing include drying, heat drying, cutting, and pulverization.

  As used herein, Gunnetum seeds refer to those composed of seed coat, thin skin, and embryo / endosperm (endosperm). As the shape and form of the Gnetsum seed, any shape and form can be used as long as the effects of the present invention can be obtained. The major axis is about 1.3 to 2.3 cm and the minor axis is about 0.6 to 1.3 cm. And those having a peanut-like shape are preferred. The Gnetum seed can be used in the present invention as long as it contains Gnetum seed, and examples thereof include Gnetum fruit which has a pericarp on the Gnetum seed.

  The processed seeds of the seeds of the present invention also include processed ones.Examples of such processed processed seeds include those in a dried state (by sun drying or the like), those in a heat-dried state, and the like. Can be In addition, as the seeds of Gnetum, those in the original state, which have not been cut and pulverized, and processed products of sliced and pulverized Gnetum seeds can be used. In the present invention, it is also possible to use an embryo / endosperm (endosperm) of a Gnetum seed or a processed product of a Gnetum seed having only a seed coat.

  In the present invention, it is desirable to use an extract of Gnetum seed, and the method for producing the extract is not particularly limited.For example, a method of extracting Gnetum seed by immersion in an immersion liquid (for example, Japan The method described in JP-A-2013-82701) can be used.

  As the immersion liquid, water, an organic solvent, or a water-containing organic solvent can be used. As the organic solvent, those which are freely miscible with water are preferable. Examples of such solvents include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol and 2-methyl-1-propanol. C1-C5 lower alcohols such as 2-methyl-2-propanol, 1-pentanol, 2-pentanol and 3-pentanol; ethers such as diethyl ether; esters such as methyl acetate and ethyl acetate. And ketones such as acetone, and organic acids such as acetic acid, glacial acetic acid, and propionic acid. The organic solvent is preferably a lower alcohol.

  The temperature of the immersion liquid at the time of immersion can be appropriately set according to the amounts of the Gnetum seed and the immersion liquid, and is, for example, 10 to 50 ° C, preferably 20 to 40 ° C. The time for immersion can be appropriately set depending on the amounts of the Gnetum seed and the immersion liquid, but is preferably 3 days or more, and more preferably 3 days to 7 days.

  The collected immersion liquid can be used as it is, but if necessary, ultrafiltration, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC), Purification can be performed by a dialysis method or a combination thereof.

  The extract of Gnetsum seeds is a recovered immersion liquid (including further purified one if necessary), a concentrated liquid obtained by concentrating the immersion liquid, and a solvent of the immersion liquid is removed by freeze-drying, spray-drying, or the like. Any state of the prepared solid can be taken. Here, concentration, freeze-drying, and spray-drying of the immersion liquid can be performed according to a conventional method. The form of the extract of Gnetum seed of the present invention is preferably in the form of a powder.

  Gunetin C is a kind of polyphenol represented by the following formula.

  Gunetin C can be used regardless of a self-prepared product or a commercially available product. Here, the method of self-preparing gonetin C is not particularly limited, and it is not limited to a method of extracting gonetin C from plants containing it, or a method of producing it by a microorganism (for example, Adil E Bala et al., “Antifungal activity of resveratrol oligomers from Cyphostemma crotalarioides ", Pesticide Science, Vol. 55, Issue 2, Pages 206-208, etc.), and a method of chemically synthesizing.

  Plants containing gnetin C are not particularly limited, and preferably include plants belonging to the family Gnetum (Ibrahim Iliya et al., “Stilbenoids from the stem of Gnetum latifolium (Gnetaceae)”, Phytochemistry, 2002 Dec; 61 (8): 959-61; Ibrahim Iliya et al., "Dimeric Stilbenes from Stem Lianas of Gnetum Africanum", HeteroCycles, VOL.57; NO.6; PAGE.1057-1062 (2002)).

  Specific examples include Gnetum latifolium, Gnetum Africanum and Gnetum gnemon (Gnetum) belonging to the family Gnetum, and are preferably Gnetum. The portion of the plant to be used is not limited as long as it contains a large amount of netin C, such as fruit (or seed), flower, leaf, etc., and preferably fruit (or seed), more preferably fruit endosperm.

  Here, when extracting netin C from a plant, the method is not particularly limited, and for example, the extraction method described above, the extraction method described in JP-A-2013-82701, and the like can be used. Gnetin C can be used either in an isolated or purified state (crude extract) or in an isolated or purified state. As the purification method, the above-described method and the like can be used.

  The purification need not be performed up to 100% purity. Gunetin C used in the present invention may have a purity of usually 50% by mass or more, preferably 80% by mass or more, and more preferably 90% by mass or more.

  In addition, when producing gnetin C using a microorganism or when acquiring it by chemical synthesis, it is preferable to carry out a purification treatment in the same manner as described above.

  Gunetin C in the present invention includes both a free state and a salt state. Examples of the salt include salts with inorganic bases such as sodium salt, potassium salt, magnesium salt, calcium salt, and aluminum salt; salts with organic bases such as methylamine, ethylamine, and ethanolamine; lysine, ornithine, arginine, and the like. Salts with basic amino acids and ammonium salts are included. The salt may be an acid addition salt. Specific examples of such a salt include mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid; formic acid, acetic acid, Organic acids such as propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid and ethanesulfonic acid; with acidic amino acids such as aspartic acid and glutamic acid Acid addition salts. In addition, hydrate, solvate, polymorph and the like thereof are also included in gnetin C in the present invention.

  The content of the enzyme-treated royal jelly in the genome stability enhancer of the present invention is not particularly limited, preferably 0.01% by mass or w / v% or more, more preferably 1.4 to 100% by mass or w / v%, and still more preferably. Is 30 to 65% by mass or w / v%.

  The content of Gnetum or an extract thereof in the genome stability enhancer of the present invention is not particularly limited, and is preferably 0.01% by mass or w / v% or more, more preferably 0.03 to 100% by mass or w / v%, More preferably, it is 30 to 65% by mass or w / v%.

  The content of gnetin C in the genome stability enhancer of the present invention is not particularly limited, preferably 0.001% by mass or w / v% or more, more preferably 0.01 to 100% by mass or w / v%, and still more preferably. It is 1 to 40% by mass or w / v%.

  The genome stability enhancer of the present invention includes cosmetics, foods and drinks (especially health foods, foods and drinks for the purpose of maintaining and promoting health (e.g., health foods, functional foods, nutritional compositions (nutritional compositions), nutritional supplements). Foods, supplements, foods for health use, foods for specified health use, nutritionally functional foods, functionally labeled foods)), quasi-drugs, and pharmaceuticals (particularly, oral medicines). The genomic stability enhancer of the present invention also includes the meaning of an additive that imparts a genomic stability enhancing effect.

  In the above-mentioned cosmetics, in addition to the above-mentioned active ingredients, components usually used in cosmetics, for example, whitening agents, humectants, antioxidants, oily components, ultraviolet absorbers, surfactants, thickeners, alcohols, Powder components, coloring materials, aqueous components, water, various skin nutrients, and the like can be appropriately compounded as needed.

  Cosmetics include any cosmetics applied to animal (including human) skin, mucous membranes, body hair, hair, scalp, nails, teeth, facial skin, lips, and the like.

  There are a wide range of cosmetic formulations, including aqueous solutions, solubilizing systems, emulsifying systems, powder systems, oil-liquid systems, gel systems, ointment systems, aerosol systems, water-oil two-layer systems, and water-oil-powder three-layer systems. Dosage forms may be employed.

  The use of cosmetics is also optional.For example, for basic cosmetics, face wash, lotion, milky lotion, cream, gel, essence, serum, pack, mask, etc. may be mentioned. Lipstick, blusher, eyeshadow, eyeliner, mascara, etc .; for nail cosmetics, manicure, base coat, top coat, remover, etc .; other, facial cleanser, (kneaded or liquid) dentifrice , Mouthwash, massage agent, cleansing agent, after shave lotion, pre-shave lotion, shaving cream, body soap, soap, shampoo, rinse, hair treatment, hair styling, hair tonic, hair restorer, antiperspirant, bath salt, etc. Is mentioned.

  In the above food and drink, in addition to the above active ingredients, if necessary, excipients, brighteners, minerals, vitamins, flavonoids, quinones, polyphenols, amino acids, nucleic acids, essential fatty acids, cooling agents , A binder, a sweetener, a disintegrant, a lubricant, a colorant, a flavor, a stabilizer, a preservative, a sustained release regulator, a surfactant, a solubilizer, a wetting agent, and the like.

  Foods and drinks include all foods and drinks that can be consumed by animals (including humans). The type of food or drink is not particularly limited.For example, beverages (soft drinks such as coffee, juice, tea drinks, milk drinks, lactic acid drinks, yogurt drinks, carbonated drinks, sakes such as sake, Western liquors, fruit wines) Spreads (such as custard cream); pastes (such as fruit paste); western confectionery (chocolate, donut, pie, cream puff, gum, jelly, candy, cookies, cakes, pudding, etc.); Japanese confectionery (such as Daifuku, mochi) , Buns, castella, anmitsu, yokan, etc.); frozen desserts (ice cream, ice candy, sherbet, etc.); foods (curry, beef bowl, porridge, miso soup, soup, meat sauce, pasta, pickles, jam, etc.); seasonings (Dressing, sprinkling, umami seasoning, soup base, etc.).

  The dosage unit form when used as a supplement is not particularly limited and can be appropriately selected, for example, tablets (e.g., uncoated tablets, sugar-coated tablets, film-coated tablets, chewable tablets, troche tablets, etc.), capsules, granules, liquids, Powders, syrups, pastes, drinks and the like can be mentioned.

  The intake of the food or drink can be appropriately set according to various conditions such as the weight, age, sex, and symptoms of the user.

  In the above-mentioned medicines, only the above-mentioned active ingredients can be used, or they can be used by mixing with other medicine ingredients described in the Japanese Pharmacopoeia such as vitamins and crude drugs.

  When the genomic stability enhancer of the present invention is prepared as a pharmaceutical, the above-mentioned active ingredient, together with a non-toxic carrier, diluent or excipient which is acceptable in pharmaceuticals, tablets (uncoated tablets, sugar-coated tablets, effervescent tablets, films) Coated tablets, chewable tablets, troches, etc.), capsules, pills, powders (powder), fine granules, granules, liquids, suspensions, emulsions, syrups, pastes, etc. Thus, an oral preparation can be obtained.

  The dose of the drug can be appropriately determined according to various conditions such as the weight, age, sex, and symptoms of the patient.

  The pharmaceuticals and cosmetics of the present invention also include quasi-drugs.

  The genomic stability enhancer of the present invention described above is applied to mammals including humans.

  The enzymatically degraded royal jelly, gnetum or an extract thereof, and gunetin C, which the genomic stability enhancer of the present invention contains as active ingredients, contain unenzymatically decomposed royal jelly and transresvera, as shown in Examples described later. The effect of inducing H2AX is higher than that of trol. Therefore, since the genomic stability enhancer of the present invention has an excellent H2AX expression inducing action, it is expected to exhibit an excellent genomic stability enhancing action.

  Since the genomic stability enhancer of the present invention has an excellent genomic stability enhancing action, it is a disease associated with genomic instability (abnormal gene repair mechanism), xeroderma pigmentosum, Huntington's disease , Werner's syndrome, Bloom's syndrome, Lynch syndrome (hereditary non-polyposis colorectal cancer) and the like, and can be suitably used for symptom reduction and treatment.

  Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited to these Examples and the like.

Enzymatic decomposition product of the material royal jelly was prepared in accordance with Example 1 of JP Japanese Patent No. 3994120. The enzymatically degraded royal jelly and the lyophilized royal jelly were dissolved in an α-MEM medium supplemented with 10% FBS so that the final concentration of the lyophilized powder was 100 μg / mL, and sterilized with a filter.

  Melinjo seed extract is obtained by immersing a crushed dried fruit of Melinjo in hydrated ethanol at room temperature as described in paragraph [0024] of JP-A-2009-013123, and concentrating the obtained extract under reduced pressure. A Melinjo seed extract containing 63.2% by mass of solid content was obtained.

  Gunetin C was obtained by subjecting 2.5 g of the above-obtained Melinjo seed extract to column chromatography (gel type: Cosmosil 75C18-PREP (manufactured by Nacalai Tesque), column size: φ3 × 26.5 cm) as eluent. Stepwise elution was carried out sequentially using aqueous methanol having a methanol content of 10% by volume, 25% by volume, 40% by volume, 80% by volume, and 100% by volume (600 mL of each eluent was flowed, and 100 mL was fractionated). When each of the obtained components was confirmed by HPLC under the following conditions, it was confirmed that gnetin C was eluted at a retention time of 33.5 minutes (a fraction eluting with water-containing methanol containing 1% by volume acetic acid and 80% by volume methanol).

<HPLC conditions>
Column: Tosoh TSKgel ODS-100V, 5 μm, 4.6 × 150 mm
Moving layer: solution A: water containing 1.0% by volume acetic acid, solution B: methanol containing 1.0% by volume acetic acid Gradient conditions: 0 minutes → 10 minutes: A: B = 65: 35 (v / v) → 63: 37 (v / v), 10 minutes → 20 minutes: A: B = 63: 37 (v / v) → 56: 44 (v / v), 20 minutes → 40 minutes: A: B = 48: 52 (v / v)
Detection wavelength: 320 nm
Flow rate: 0.8 mL / min

  After the above fractions were concentrated, they were sequentially eluted by medium pressure column chromatography under the following conditions.

<Medium pressure column chromatography conditions>
Gel type: Silica gel, Daisogel IR-60-40 / 63-W
Column size: φ2 x 7.48 cm
Detection wavelength: 320 nm
Moving layer: solution A: methanol, solution B: chloroform gradient Conditions: 0 minutes → 2 minutes: A: B = 11: 89 (v / v), 2 minutes → 8 minutes: A: B = 11: 89 (v / v) → 18: 82 (v / v), 8 minutes → 12 minutes: A: B = 18: 82 (v / v), 12 minutes → 14 minutes: A: B = 21: 79 (v / v) , 14 minutes → 20 minutes: A: B = 21:79 (v / v) → 29:71 (v / v), 20 minutes → 24 minutes: A: B = 29:71 (v / v), 24 minutes → 30 minutes: A: B = 29:71 (v / v) → 36:64 (v / v), 30 minutes → 34 minutes: 36:64 (v / v)
Flow rate: 60 ml / min

  The eluate was collected in 60 mL portions, and the 29th to 30th fractions, which showed an absorption peak at a detection wavelength of 320 nm, were concentrated using an evaporator to obtain netin C (35.2 mg, purity: 97%).

  Trans resveratrol was obtained from Sigma-Aldrich.

Test example 1
Growth arrest of normal cells is induced by a decrease in the level of H2AX (essential for inducing DNA damage repair factors to accumulate at DNA damage sites), and the DNA repair ability is also reduced. Genomic instability has been shown to be induced in this context due to growth stimuli. Therefore, the effects of enzymatically degraded royal jelly (enzyme-treated RJ) and royal jelly (untreated RJ) on H2AX expression were analyzed.

MEF (mouse embryo fibroblast) cells were cultured in a DMEM medium containing 10% FCS (fetal calf serum) supplemented with 25 μg / ml enzyme-treated RJ or untreated RJ at 37 ° C. under 5% CO ₂ under the 3T3 method. And cultured. Cells cultured for 0, 3, 6, 12, and 24 hours were collected, and subjected to Western analysis using an H2AX antibody (Bethyl Laboratories, Inc.) and a β-actin antibody (AC-74, Sigma-Aldrich).

  The results of the Western analysis and the results of the Western analysis were obtained using ImageJ (Rasband, WS, ImageJ, US National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2016.). FIG. 1 shows a graph quantified by using the graph. Transient induction of H2AX expression was found for both enzymatically degraded RJ and untreated RJ treatment. Compared with lane 2, the expression amount of H2AX was 2.1 times the amount of lane 8. This result indicates that the enzyme-degraded RJ has a transient expression inducing capacity of 2.1 times that of the untreated RJ.

Test example 2
Using the same method as in Test Example 1, the effect of Melinjo and transresveratrol on H2AX expression was analyzed. In this case, 0.26, 0.52, 1.0, 2.1 w / v% melinjo seed extract or transresveratrol was added to the medium, and cells cultured for 1 hour were collected.

  FIG. 2 shows a result of the Western analysis and a graph obtained by digitizing the result of the Western analysis. As a result, both Melinjo and trans-resveratrol found transient induction of H2AX expression. Compared with Lane 4, Lane 9 showed a 2.7-fold increase in H2AX expression. This result indicates that Melinjo has a 2.7-fold ability to induce transient expression of H2AX as compared to transresveratrol.

Test example 3
Using the same method as in Test Example 1, the effects of transresveratrol, melinjo and gunetin C on H2AX expression were analyzed. In this case, 2.5 μM (0.52 μg / ml) transresveratrol, 0.52 μg / ml melinjo seed extract, or 2.5 μM gunetin C was added to the medium, and 0, 1.5, 3, 6, 12, The cells cultured for 24 hours were collected.

  The results of the Western analysis and the results of the Western analysis were obtained using ImageJ (Rasband, WS, ImageJ, US National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2016.). FIG. 3 shows a graph quantified by using the graph. As a result, resveratrol, merinjo, and gunetin C were all found to transiently induce H2AX expression. Melinjo (lane 4) and gunetin C (lane 4) had 1.9-fold and 1.8-fold higher H2AX expression levels than trans-resveratrol (lane 3), respectively. This result indicates that Merinjo and Gunetin C have 1.9 times and 1.8 times the ability to induce transient expression of H2AX as compared with transresveratrol.

Test example 4
To examine the effect of Melinjo on enhancing genomic stability in vivo, Msh2-KO mice lacking a mismatch repair factor {Lynch syndrome model that produces microsatellite instability (MSI), a type of genomic instability} Experiments. It is known that the mice die from about 10 months of age and about half a year.

  FIG. 4 shows the results. As a result of comparing the group (■) administered with 0.3% by mass (360 mg / kg body weight / day) of a Melinjo seed extract capable of inducing transient expression of H2AX (■) and the group fed with a normal diet (◆), More than half of the group on the normal diet died by 20 weeks, while the group on the diet containing the Melinjo seed extract did not die during this period.

  This clearly shows that when the gene repair function is reduced, the "genomic stability enhancer" having a transient H2AX expression-inducing activity produces an "effect of extending life span".

Test example 5
We examined whether taking Melinjo had a preventive effect. Msh2-KO mice of 8-12 weeks of age were administered a 0.03 w / v% melinjo seed extract group (36 mg / kg body weight / day), or as a control group, mice administered sterile water were treated with a carcinogen. Water containing some potassium bromate (0.02%) was provided as drinking water. After 24 weeks, the number of tumors formed in the intestinal tract was counted, and the Melinjo group and the control group were compared. Msh2-KO mice deficient in mismatch repair factor are prone to genomic instability, and tumors are easily formed in the digestive tract by BrKO ₃ , a chemical carcinogen.

  FIG. 5 shows the results. In the group to which the Melinjo seed extract was administered, a clear inhibitory effect on the carcinogenic level was observed. This indicates that even in a constitution in which genomic instability is likely to occur, it is possible to prevent the onset of cancer by taking the Melinjo seed extract prophylactically.

Claims (2)

  A genomic stability enhancer comprising as an active ingredient at least one selected from the group consisting of enzymatically degraded royal jelly, gnetum or an extract thereof, and gnetin C.   The genome stability enhancer according to claim 1, which is a cosmetic, a food or drink, a pharmaceutical, or a quasi-drug.

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